Substandard and Falsified Antibiotics and Medicines against Noncommunicable Diseases in Western Cameroon and Northeastern Democratic Republic of Congo.

Falsified and substandard medicines may undermine the progress toward the Sustainable Development Goals. The present study investigated the quality of 13 essential medicines in Cameroon and the Democratic Republic of Congo (DR Congo). Five hundred six medicine samples were collected from the government and faith-based health facilities, private pharmacies, and informal vendors (total 60 facilities). Collected samples were analyzed according to the U.S. Pharmacopeia (USP) for identity, content, and dissolution of their active pharmaceutical ingredients (APIs) and for uniformity of dosage units. Three samples (0.6%) were identified as falsified. Overall, 8.5% of the samples failed USP specifications for the content of the API and 11.7% failed dissolution testing. Medicines from informal vendors showed a higher out-of-specification rate (28.2%) than other types of drug outlets (12.3%; P < 0.0001). All three falsified medicines had been sold by informal vendors. The failure rate of medicines stated to be produced in Europe (5.1%) was lower than that for medicines from Asia (17.7%; P = 0.0049) and Africa (22.2%; P = 0.0042). Medicines against noncommunicable diseases showed a higher failure rate than antibiotics (25.3% versus 12.1%; P = 0.0004). Four hundred fifty-one of the samples were analyzed in Cameroon and the DR Congo with the Global Pharma Health Fund Minilab (thin-layer chromatography and disintegration testing). The three falsified medicines were readily detected in Minilab analysis. However, substandard samples were detected with low sensitivity. A well-enforced ban of medicine sales by informal vendors and increased attention to supplier qualification in the procurement process may reduce the prevalence of substandard and falsified medicines.

Identification of Falsified Chloroquine Tablets in Africa at the Time of the COVID-19 Pandemic.

Reports that chloroquine and hydroxychloroquine may be effective against COVID-19 have received worldwide attention, increasing the risk of the introduction of falsified versions of these medicines. Five different types of falsified chloroquine tablets were discovered between March 31, 2020 and April 4, 2020, in Cameroon and the Democratic Republic of Congo by locally conducted thin layer chromatographic analysis. Subsequent investigation by liquid chromatography and mass spectrometry in Germany proved the absence of detectable amounts of chloroquine and the presence of undeclared active pharmaceutical ingredients, that is, paracetamol and metronidazole, in four of the samples. The fifth sample contained chloroquine, but only 22% of the declared amount. Such products represent a serious risk to patients. Their occurrence exemplifies that once medicines or vaccines against COVID-19 may be developed, falsified products will enter the market immediately, especially in low- and middle-income countries (LMICs). Timely preparations for the detection of such products are required, including the establishment of appropriate screening technologies in LMICs.

Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus.

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent.

Cyclodextrin-mediated enantioseparations by capillary electrochromatography.

Immobilized cyclodextrin derivatives are used as chiral selectors in various modes of capillary electrochromatography (CEC). The present chapter describes three techniques in detail utilizing CDs in CEC: (1) open-tubular capillary electrochromatography (o-CEC), (2) packed capillary electrochromatography (p-CEC), and (3) monolithic capillary electrochromatography (rod-CEC). Nanoparticle pseudostationary phase capillary electrochromatography (psp-CEC) is briefly discussed.

Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147.

Ltnα and Ltnß are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.

Myristate is selectively incorporated into surfactant and decreases dipalmitoylphosphatidylcholine without functional impairment.

Lung surfactant mainly comprises phosphatidylcholines (PC), together with phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75-80% of surfactant PC. During alveolarization, which occurs postnatally in the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/16:0. As lipoproteins modify surfactant metabolism, we postulated an extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We, therefore, fed rats (d19-26) with trilaurin (C12:0(3)), trimyristin (C14:0(3)), tripalmitin (C16:0(3)), triolein (C18:1(3)) or trilinolein (C18:2(3)) vs. carbohydrate diet to assess their effects on surfactant PC composition and surface tension function using a captive bubble surfactometer. Metabolism was assessed with deuterated C12:0 (ω-d(3)-C12:0) and ω-d(3)-C14:0. C14:0(3) increased PC16:0/14:0 in surfactant from 12 ± 1 to 45 ± 3% and decreased PC16:0/16:0 from 47 ± 1 to 29 ± 2%, with no impairment of surface tension function. Combined phospholipase A(2) assay and mass spectrometry revealed that 50% of the PC16:0/14:0 peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). While C12:0(3) was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:0(3), C18:1(3), and C18:2(3) had no significant effect on PC16:0/16:0 or PC16:0/14:0. d(3)-C14:0 was enriched in lung PC, either via direct supply or via d(3)-C12:0 elongation. Enrichment of d(3)-C14:0 in surfactant PC contrasted its rapid turnover in plasma and liver PC, where its elongation product d(3)-C16:0 surmounted d(3)-C14:0. In summary, high surfactant PC16:0/14:0 during lung development correlates with C14:0 and C12:0 supply via specific C14:0 enrichment into lung PC. Surfactant that is high in PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal respiration and surfactant function in vitro.

Chiral silica-based monoliths in chromatography and capillary electrochromatography.

Chiral-modified silica-based monoliths have become well-established stationary phases for both high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The silica-based monoliths were fabricated either in situ in the capillaries for nano-HPLC and CEC or in a mould for "conventional" HPLC. The present review summarizes the chiral modification of silica monoliths and the recent development in the field of enantioselective separations by nano-HPLC and CEC.

Delta-cyclodextrin as novel chiral probe for enantiomeric separation by electromigration methods.

Native delta-CD has been employed as chiral selector in CE and MEKC. To investigate the potential of the enantiodiscriminating properties of delta-CD, negatively charged 5-dimethylamino-1-naphthalene-sulfonyl (dansyl)-, 2,4-dinitrophenyl (DNP)- and FMOC-derivatives of several amino acids, 1,1'-binaphthyl-2,2'-diylhydrogenphosphate, flavanones and three positively charged drugs have been selected as testing samples. Enantioresolution factors up to 4.82 have been observed. The results were compared with those achieved by the conventional running buffer additives alpha-, beta- and gamma-CDs. For several examples a steady increase of enantioresolution with increasing degree of oligomerization has been detected.

Comparison of monolithic approaches for enantioselective capillary electrochromatography involving cyclodextrins.

The access to CD-modified monoliths for enantiomeric separation by CEC can be divided into two main approaches. (i) Silica-based monoliths, prepared by either a sol-gel process or by sintering of silica particles, are modified after fabrication by coating with a CD selector. Alternatively the fusion of CD functionalized silica particle via gluing is feasible. (ii) Rigid or homogeneous organic polymer-based monoliths, prepared by polymerization of organic monomers in the presence of a porogen, are modified with the CD selector either by copolymerization or by physical incorporation into the continuous bed.

Stereoisomeric separation of flavanones and flavanone-7-O-glycosides by capillary electrophoresis and determination of interconversion barriers.

The stereoisomeric separation of several flavanones and flavanone-7-O-glycosides has been achieved with capillary electrophoresis by adding native cyclodextrins or cyclodextrin derivatives to the background electrolyte. As an alternative method, micellar electrokinetic chromatography with sodium cholate as a chiral surfactant has been used for the epimeric separation of two flavanone-7-O-glycosides. The effect of buffer systems containing mixtures of cyclodextrin with either sodium dodecyl sulfate or sodium cholate upon the chiral recognition of flavanones and flavanone-7-O-glycosides as well as the variation of the background electrolyte (concentration of buffer and surfactant, pH value, organic modifier), and its influence on the resolution factor Rs was investigated. Temperature- and pH-dependent enantiomerization or epimerization barriers of several flavanones (naringenin, homoeriodictyol) and flavanone-7-O-glycosides (naringin, neohesperidin, prunin, narirutin) in basic media (pH values of 9-11) have been observed. Interconversion profiles featuring characteristic plateau formation of the elution pattern were observed at high pH and evaluated with the simulation software ChromWin to determine rate constants k(T) and Eyring activation parameters, DeltaG#(T), DeltaH#, and DeltaS#.

Enantiomeric separation by capillary electrochromatography using monolithic capillaries with sol-gel-glued cyclodextrin-modified silica particles.

By an on-column sol-gel process, a chiral monolithic stationary phase was prepared by the fusion of permethyl-beta-cyclodextrin-silica (Chira-Dex-silica) particles and by linking them to the internal capillary wall. The resulting monolith is stable toward voltage (30 kV) and pressure (300 bar) and possesses a high efficiency (up to 100,000 theoretical plates per meter). Efficient enantiomeric separation of various chiral compounds by pressure-supported capillary electrochromatography (CEC) was achieved. When comparing this method to capillary liquid chromatography (LC) employing the same column in an unified equipment, CEC shows a twofold higher column efficiency at comparable elution times and hence better resolution factors.

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique.

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.

Recent progress in enantiomeric separation by capillary electrochromatography.

Recent progress in enantiomeric separations by capillary electrochromatography (CEC) is reviewed. The development of simple and robust CEC column technologies plays an important role for popularization of CEC. During the last several years, various approaches for the preparation of enantioselective columns have been reported. Currently, the monolithic column technology (continuous beds) represents the most advanced approach for the preparation of CEC columns. The development of new chiral stationary phase used for CEC is another important issue in this field. Fundamental investigations on electrochromatographic behaviors of various CSPs are necessary in order to understand the separation mechanism and thus improve the separation performance. Some chiral stationary phases performed better under nonaqueous CEC conditions than reversed-phase conditions. Coupling CEC with mass spectrometry (MS) provides a powerful tool for enantiomeric separation. Finally, some applications of enantiomeric separation by CEC are summarized.

On-line coupling of packed capillary electrochromatography with coordination ion spray-mass spectrometry for the separation of enantiomers.

Pressure-supported packed capillary electrochromatography (CEC) and packed capillary high-performance liquid chromatography (pHPLC) have been coupled on-line to electrospray ionization-mass spectrometry (ESI-MS) and coordination ion spray-mass spectrometry (CIS-MS). Separation of enantiomers of barbiturates and chlorinated alkyl phenoxypropanoates were performed on a permethylated beta-cyclodextrin stationary phase by pressure-supported CEC. For on-line detection with ESI- and CIS-MS, a modified sheath-liquid interface was used. CIS-MS is a universal, novel ionization technique which improves the selectivity as well as the sensitivity. Charged complexes were formed through the addition of central complexing ions such as silver(I), cobalt(II), copper(II), and lithium(I) to the sheath flow. Advantages of CIS-MS detection compared to the ESI-MS mode are discussed. In the CIS-MS mode, increased sensitivity and high selectivity was attained through different possibilities of complexation. The superiority of pressure-supported CEC compared to pHPLC in the hyphenation with CIS-MS is demonstrated.

A silica monolithic column prepared by the sol-gel process for enantiomeric separation by capillary electrochromatography.

A method for the preparation of a silica monolithic capillary electrochromatography (CEC) column for the separation of enantiomers has been developed. The porous silica monolith was fabricated inside a fused-silica capillary column by using the sol-gel process. After gelation for 24 h, hydrothermal treatment at 100 degrees C for 24 h was performed to prevent the sol-gel matrix from cracking. The prepared monolith was then coated with Chirasil-beta-Dex which represents a chiral polymer prepared by grafting permethyl-beta-cyclodextrin to polymethylsiloxane with an octamethylene spacer. Immobilization of Chirasil-beta-Dex was performed by heat treatment at 120 degrees C for 48 h to give a nonextractable coating. The column performance was evaluated by using racemic hexobarbital as a model compound. The efficiency of 9.2 x 10(4) theoretical plates/m for the first eluted enantiomer of hexobarbital was obtained at an optimal flow rate of the mobile phase. The effect of mobile phase composition on enantiomeric separation of hexobarbital was also investigated. The column proved to be stable for more than one hundreds of runs during a two-months period. The enantiomers of several neutral and negatively charged chiral compounds were baseline separated on this column.